J. Cell Sci. 47. 267-275 (1981)
Printed in Great Britain © Company of Biologists Limited
FUNCTIONAL STATES OF RNA POLYMERASE
IN THE MACRONUCLEUS OF TETRAHYMENA
PYRIFORMIS AND THEIR DEPENDENCE ON
CULTURE GROWTH
MANFRED FREIBURG
Zoologisclies Institut der Universitdt Miinster, Badestr. 9 D-4400 Milnster, FRG
SUMMARY
RNA polymerase I in macronuclei of late log-phase cells of the ciliate Tetrahymena pyriformis
is found to be present in 2 functional states, the one being actively engaged in transcription of
nbo8omal genes, the other one in a ' free' state, i.e. free to transcribe added DNA or poly d(AT).
Stimulation of RNA synthesis induced by dilution of stationary-phase cells into fresh medium
is correlated with an increase in activity of template engaged and a decrease of free RNA
polymerase I.
In contrast, RNA polymerase II shows no pronounced changes in activity and is not available
to transcribe poly d(AT). The data favour the assumption that factors other than the amount of
RNA polymerase I molecules available control transcription of the ribosomal genes.
INTRODUCTION
The ciliate Tetrahymena pyriformis provides an excellent system to investigate the
regulation of genes coding for ribosomal RNA because changes in the culture conditions, such as inoculation of a stationary-phase culture into fresh medium result in a
rapid increase of rRNA synthesis (Cameron & Guile, 1965; Andersen & Nielsen,
•979)- Incorporation studies employing whole cells are difficult to interpret because
many parameters such as stability, uptake and metabolism of the RNA precursors
influence the experimental results (Jauker & Hipke, 1975). On the other hand,
investigations using an in vitro transcription system of Tetrahymena did not yield any
evidence of a specific interaction of purified RNA polymerase with macronuclear DNA
(Freiburg, 1977). Therefore I have tried a third approach by employing isolated
macronuclei. The results presented here show that they represent a promising
experimental system, already previously introduced for the study of transcription in
Tetrahymena by Mita, Shiomi & Iwai (1966) and Lee & Byfield (1970). In isolated
macronuclei problems concerning uptake and differences in pool sizes of precursors
are negligible and the different classes of RNA polymerases within the nuclei can easily
be distinguished by their different sensitivity to the mushroom poison a-amanitin.
There is RNA polymerase I, the a-amanitin-insensitive enzyme which transcribes the
ribosomal genes, the very sensitive RNA polymerase II which synthesizes the heterogenous nuclear RNA, and the intermediately sensitive RNA polymerase III transcribing the tRNA and 5s RNA genes (for review, see Roeder, 1976). Moreover,
268
M. Freiburg
isolated macronuclei are not as complex as whole cells, but still retain some of those
characteristics which reflect the situation within the cell. Attempts will be reported to
distinguish different functional states of RNA polymerase I which are related to
gene activity.
MATERIALS AND METHODS
Tetrahymena pyriformis strain T (amicronucleate) was grown in 0-75 % proteose peptone
no. 3 (Difco), 075 % yeast extract (Difco), 1-5 % glucose, 1 mM MgSO 4 , 0-05 mM CaCl 2 and
0003 % sequestrene at 29 °C in a New Brunswick shaker at 125 rev/min. For the isolation of
macronuclei cells from 600 ml were harvested, if not otherwise indicated, during the late
logarithmic growth phase (approx. 12 x 10' cells/ml) and resuspended in 50 ml T C M buffer
(10 mM Tris-HCl, pH 7-9, 3 mM CaCl 2) 1 mM MgCl 2 ) containing 025 M sucrose, 0-2%
Nonidet P 40, o-i % spermidine and 35 /tg phenylmethylsulphonylfluoride (PhMSF, dissolved
in ethanol, added to buffers immediately before use). The cells were lysed with 2-3 strokes of a
tight pestle in a Teflon-glass homogenizer. The lysate was centrifuged for 10 min at 1000 g, and
the resulting pellet resuspended in 130 ml T C M buffer containing 1-75 M sucrose, o-i %
spermidine and g fig PhMSF. The suspension was layered in 3 portions on 10-ml cushions of
T C M buffer containing 1-75 M sucrose, and centrifuged for 40 min at 20000 rev/min in a SW
25 2 rotor. The purified macronuclei at the bottom of the tube were resuspended in 50 mM
Tris-HCl, pH 7 9 , 3 0 % glycerol, 5 mM MgCl 2 , 1 mM EDTA end 0 5 mM dithiothreitol,
frozen in liquid nitrogen and stored at —70 °C. The macronuclei were free of cytoplasmic
contaminants as checked in the phase-contrast microscope. The isolation efficiency was
approx. 90 %. All steps were carried out at 4 °C. DNA was puiined from isolated macronuclei
following the method given by Gross-Bellard, Oudet & Chambon (1973) and denatured by
heating for 10 min in boiling water and subsequent rapidly chilling in ice. The native DNA had
an average molecular weight of 3-65 x 10' (Freiburg, 1977). For measurement of RNA synthesis in a growing culture aliquots were taken at various times and incubated for 30 min at 29 °C
with 5 /iCi/ml [ 3 H]uridine (30 Ci/mmol). The reaction was stopped by adding an equal volume
of 50 mM Tris-HCl, pH 7 - 9 , 1 % SDS, o-1 M NaCl and 5 mM EDTA and subsequent incubation
for 1 min in boiling water. After precipitation with ice-cold 0-5 M TCA the samples were filtered
through Sartorius glass fibre filters (type 13400) and washed with 001 M TCA. The radioactivity on the dried filters was determined by liquid scintillation spectrophotometry.
RNA synthesis in isolated macronuclei was measured by incubation of 20 fi\ of the nuclear
suspension in a final volume of 13s /*1 containing 50 mM Tris-HCl, pH 7 9 , 2 mM ATP, 1 mM
each of C T P and G T P (if not indicated otherwise), 0 1 mM U T P , 0 7 fid 3 H - U T P (10 Ci/
mmol), o-i M KC1 and 3 mM MnCl,. As an exogenous template denatured Tetrahymena
macronuclear DNA (56 /tg/ml) or poly d(AT) (80 /tg/ml) were used. The choice of these
templates appeared to be of advantage for a general test of RNA polymerase activity since
specific initiation is no prerequisite for transcription of these templates. RNA polymerase II was
measured as that fraction of total RNA polymerase activity which can be inhibited by the addition of 8 /tg/ml a-amanitin/test, whereas the remaining insensitive fraction represents the
activity of RNA polymerase I (III). The reactions were usually carried out for 30 min at
29 °C, stopped by the addition of 2 ml ice-cold carrier RNA (50 /fg/ml in 2 M NaCl) and
0 5 ml of 3 M TCA and treated as indicated above. Each value represents the average of 4
parallel determinations.
RNA polymerase was purified from isolated macronuclei according to Roeder & Rutter
(1969) except that the enzyme was eluted from DEAE-Sephadex A-25 in a i-step procedure
without separating different species.
RESULTS
RNA synthesis in isolated macronuclei
To discriminate between the activities of RNA polymerases I, II and III in
isolated macronuclei, the dose-dependent inhibition of RNA synthesis by a-amanitin
has been tested (Fig. 1). The initial rapid inhibition by low doses is due to the inhibi-
RNA polymerase in Tetrahymena
269
100O
5 10
50
100
500
Q-amanitin, /jg/ml
Fig. 1. Dose-dependent inhibition of RNA synthesis by a-amanitin in isolated
macronuclei.
tion of RNA polymerase II (Roeder, 1976), the remaining activity represents the sum
of the activity of RNA polymerases I and III. Since the inhibition observed by an
increase of the a-amanitin concentration from 10 to ioo/tg/ml clearly exceeds that
caused by an increase from 100 to 5oo/6g/ml, it may be suggested that the fraction
of intermediate sensitivity corresponds to RNA polymerase III. Similar characteristics
have been reported for RNA polymerase III from other objects (Roeder, 1976).
The incorporation of 3 H-UMP into RNA by RNA polymerase I (III) and II in
isolated macronuclei is demonstrated in Fig. 2 as a function of time. The rate of
3
H-UMP incorporation using endogenous DNA as template shows a gradual decline;
there is only a very slight residual increase of incorporation into RNA after an incubation time of 15 min. This reflects a characteristic feature of in vitro transcription in
isolated macronuclei: those RNA chains which have been initiated in vivo, are elongated and terminated in vitro without measurable reinitiation. This conclusion is
supported by the finding that heparin which prevents initiation of free RNA polymerase molecules already at low doses, has no effect on the transcription in isolated
macronuclei (Table 1).
The addition of denatured macronuclear DNA to isolated macronuclei strongly
stimulates the activity of RNA polymerase I (III), while the activity of RNA polymerase II remains nearly unaffected (Fig. 2). After an incubation time of 15 min,
when the transcription of the endogenous template ceases, the rate of DNA-stimulated
RNA synthesis by RNA polymerase I (III) is still very high. It appears that the DNA
added to macronuclei is essentially not transcribed by RNA polymerase molecules
released from endogenous transcription complexes after termination, because a high
proportion of RNA polymerase transcribes the exogenous DNA already at the start of
the reaction, when most of the molecules engaged in transcribing the endogenous
template have not yet terminated.
Template-engaged and 'free' RNA polymerase molecules in the macronucleus
The finding that denatured DNA added to isolated late log-phase macronuclei
stimulates preferentially the activity of RNA polymerase I (III), gives rise to the
M. Freiburg
270
10
o
Q.
O
o
c
i
40
15
Incubation time, mm
Fig. 2. Incorporation of ' H - U M P into RNA in isolated macronuclei as a function of
time. Activity of RNA polymerase I (III) ( O
O) and RNA polymerase II
(A
A) without added DNA; activity of RNA polymerase I (III) ( •
•)
and RNA polymerase II (A
A) with added denatured Tetrahymena DNA.
Each reaction mixture contained 2 8 x 10* macronuclei.
Table 1. Effect of heparin on the total 3H- UMP incorporation in isolated macronuclei
and on the transcription of native Tetrahymena DNA by purified RNA polymerase
Heparin,
/ig/ml
Macronuclei,
cpm
2
481 ± 8
474 ± 57
8
456 ±59
0
RNA polymerase + DNA,
cpm
8549 ±511
64 ± 19
Tests were carried out as described in Materials and Methods with
isolated macronuclei (8'1 x io 6 per test) or purified RNA polymerase
(20 y\ per test, o.D.jgj = 115) and native Tetrahymena DNA (26 /tg
per test) in the presence of different concentrations of heparin. The
results are expressed as average values of 4 determinations and the
corresponding standard deviation.
assumption that there are 2 different states of RNA polymerase I (III) within these
macronuclei, one of which being engaged in transcription, the other existing in a ' free'
form, i.e. free to transcribe added DNA. (Presumably this RNA polymerase is not
part of an active ternary transcription complex before DNA is added.) The activity of
'free' RNA polymerase was measured by the transcription of added denatured
Tetrahymena DNA or by transcription of poly d(AT) and compared with the activity
of the template-engaged enzyme (Table 2). Using the endogenous DNA as template,
RNA polymerase in Tetrahymena
271
Table 2. Template-engaged and free RNA polymerase molecules in isolated macronuclei
Substrates
ATP
ATP
CTP
GTP
'H-UTP
'H-UTP
A
1
"^--^^^
Template . . .
Enzyme^^--^^^
activity
~~~--~^^^
RNA polymerase I (III), cpm
RNA polymerase II, cpm
Endog
DNA
1289
1016
1
Endog. DNA
+ denatured
DNA
Endog.
DNA
Endog. DNA
+
poly d(AT)
7796
79
5224
1543
0
0
Using endogenous DNA as template and the 4 nucleoside triphosphates all of the RNA
polymerase molecules being in a specific transcription complex are active. Addition of poly
d(AT) and the 2 nucleoside triphosphates ATP and UTP leads to ribo AU synthesis, which is
completely dependent on the exogenous template and is catalysed by RNA polymerase molecules existing in a free state.
the activities of RNA polymerase I (III) and II are present in a ratio of 1:1. The
addition of denatured DNA stimulates the a-amanitin-insensitive fraction approx.
6-fold, while the activity of RNA polymerase II increases only slightly compared to
the stimulation of RNA polymerase I (III). Omission of the 2 nucleoside triphosphates CTP and GTP inhibits the transcription of the endogenous template almost
completely, whereas addition of poly d(AT) leads to a ribo AU synthesis by RNA
polymerase molecules which are not template-engaged and exclusively of the
a-amanitin-resistant type.
Functional states of RNA polymerase during culture growth
Changes in transcriptional activities can be induced experimentally either by
starvation and refeeding of Tetrahymena cultures (Hallberg & Bruns, 1976; Nilsson,
1976) or by dilution of stationary phase cultures into fresh medium (Cameron & Guile,
1965; Conner & Koroly, 1973; Andersen & Nielsen, 1979). The latter method was
chosen to test the effect of different growth characteristics on the functional states of
RNA polymerase.
A Tetrahymena culture was grown up in 300 ml culture medium to a cell density of
io6 cells/ml, and harvested after further incubation for 14 h, when cells have reached
the stationary phase. To start new culture growth, cells were then inoculated into 900
ml fresh prewarmed medium. Aliquots of the culture were taken at the indicated
times and the incorporation of [3H]uridine/cell and the cell number determined
(Fig. 3 A). One hour after inoculation into'fresh medium, the incorporation of [3H]uridine/cell was increased approx. 4-fold, whereas cell multiplication had not yet been
started. With the beginning of the exponential growth after 2 h the [3H]uridine incorporation reached its maximum and declined during the following exponential
M. Freiburg
272
0
1
2
3
4
6
Time after inoculation into fresh medium, h
Fig. 3. Culture growth-dependent changes in RNA synthesis and the state of RNA
polymerase after inoculation of a stationary culture into fresh medium. A. Cell number
(O
O) and ['H]uridine incorporation/cell (x
x ) . u. Culture growthdependent free and template-engaged RNA polymerases in isolated macronuclei.
Parallel cultures were started by inoculation of a stationary culture into fresh medium.
At different times after inoculation macronuclei were isolated and assayed for free and
template-engaged RNA polymerase activities. Template-engaged RNA polymerase I
(III) (O
O) and RNA polymerase II (A
A) active on the endogenous
DNA. Free RNA polymerase I (III) ( •
• ) active on added poly d(AT) and
ATP and UTP as substrates only.
growth. Already 4 h after inoculation into fresh medium the cells again reached the
transition phase between exponential and stationary phase.
In a similar experiment macronuclei were isolated at different times after the start
of a new culture growth, and the activities of free and template-engaged RNA
polymerase were determined (Fig. 3B). Two hours after inoculation the activity of
bound RNA polymerase I was found to reach a maximum and declined during further
RNA polymerase in Tetrahymena
273
3
incubation. In this respect it shows a similar time course to the [ H]uridine incorporation in whole cells. The activity of free RNA polymerase I decreases immediately
while the activity of bound RNA polymerase I increases. In contrast, the decrease of
bound RNA polymerase I activity following maximum activation is not paralleled by
an increase of the free enzyme, but shows a time lag, in this case about 3 h.
DISCUSSION
The data presented in this communication show that as in other eucaryotes (for
review, see Muramatsu, Matsui, Onishi & Mishima, 1979; Grumint, 1978) isolated
macronuclei of the ciliate Tetrahymena pyriformis contain a pool of RNA polymerase I
molecules which are - depending on the actual culture growth - either actively
transcribing the ribosomal genes or existing in a free state, i.e. not bound to DNA.
The finding that a rapid increase of RNA synthesis is correlated with the reduction of
the activity of free enzyme favours the assumption that the factor limiting transcription
of the ribosomal genes is not the number of RNA polymerase molecules available but
their ability to initiate rRNA synthesis. There is increasing evidence that the synthesis
of rRNA in eucaryotes is stringently linked to protein synthesis, whereby initiation by
RNA polymerase I is positively controlled by a short-lived protein which enables the
free enzyme to bind to specific sites of the DNA and to initiate new RNA chains
(Gross & Pogo, 1976; Mishima, Matsui & Muramatsu, 1979). Perhaps the time lag
between the decrease in activity ot bound RNA polymerase I and the increase in the
free enzyme indicates that there may be a second mechanism of gene regulation causing
direct repression of transcription. Such a mechanism is known from Physarum where
free RNA polymerase I is inactivated under conditions of starvation by a specific
inhibitor which disappears after refeeding (Hildebrandt & Sauer, 19776; Hildebrandt,
Mengel & Sauer, 1979). The finding that a-amanitin-sensitive RNA polymerase II
cannot be stimulated by addition of exogenous DNA or poly d(AT) to isolated
macronuclei leads to the conclusion that - in contrast to RNA polymerase I - this
type of enzyme remains always bound to its template. Dreyer & Hausen (1978) also
reported that RNA polymerase II is always bound to DNA in lysates of Ehrlich ascites
cells. In contrast, Hildebrandt & Sauer (1977a) in Physarum, and Yu (1974, 1975) in
rat liver have found high amounts of free RNA polymerase II in isolated nuclei.
These conflicting results may be due to different isolation and assay procedures or
specific features of the different organisms used in the experiments. A selective loss of
free RNA polymerase II in the Tetrahymena system can be excluded by the finding
that RNA polymerase isolated from whole cells shows an elution profile on DEAESephadex columns similar to that of enzyme preparations from isolated macronuclei
(unpublished results).
Furthermore, RNA polymerase II shows no pronounced changes in its activity after
inoculation of stationary phase cells into fresh medium. Similar results are reported
from other eucaryotic systems after stimulation of RNA polymerase I (Todthunter,
Weissbach & Brot, 1978; Chomczynski, Sok61-Misiak & Kleczkowska, 1977; Cox,
1976).
274
M. Freiburg
I want to thank Dr Karl Muller for helpful discussions and critical reading of the manuscript.
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{Received 23 June 1980)